Viral safety is a basic requirement for therapeutic proteins, especially for plasma-derived drugs. Over the recent years, substantial progress in virus safety had been made by almost eliminating the risk of virus transmission. Specific steps designed to remove or inactivate viruses were developed, validated and introduced in the manufacturing process of therapeutically used plasma proteins. The manufacturing processes of coagulation factors like factor VIII or factor IX, plasma proteinase inhibitors such as antithrombin, antitrypsin or C1-inhibitor, and of albumin and immunoglobulins must have at least one inactivation step effective against lipid-enveloped viruses to fulfill the safety requirement, and national and international regulatory bodies have set up a comprehensive framework of regulations to ensure maintenance and further improvement of virus safety. For example, the scientific Committee for Proprietary Medicinal Products (CPMP) as an organ of the European Medicines Agency (EMA) issued guidelines covering virus safety of biologicals. To minimize the risk of virus transmission by plasma-derived drugs, CPMP guidelines besides other preventive actions on the level of plasma as the starting material, strongly recommend the incorporation of two independently acting virus inactivation procedures in the manufacturing process.
The knowledge of the usefulness of detergents for viral inactivation together with the availability of chromatographic protein purification methods has shown the way to viral inactivation by detergents in plasma protein processing. The primary target viruses HIV, HBV, and HCV are all lipid-enveloped. Therefore, an effective virus inactivation strategy rationally takes advantage of viral susceptibility to detergent alone or to the well established solvent-detergent (S/D) procedure. The effect of all these procedures lies in the specific disruption of the viral lipid envelope, while having a relatively low impact on the integrity of therapeutic proteins with the exception of lipoproteins.
The non-ionic detergent polyoxyethylene sorbitan monooleate (polysorbate 80, Tween™ 80) is the most commonly used virucidal detergent of the polysorbates. Following the virus inactivation step, the manufacturing process has to comprise a purification step effective to remove the inactivation agents, such as chromatographic adsorption of the protein. In order to monitor the efficiency of the detergent removal procedure and also to check the compliance with the specified and validated limits of detergent concentration during the virus inactivation, however, the determination of the detergent has to be performed in the presence of varying and occasionally high concentrations of proteins, e.g. plasma proteins. The chemical composition and the structural diversity of polysorbates like polysorbate 80, the low reactivity of these compounds, the protein/buffer matrix as well as the possible presence of other physico-chemically related detergents such as e.g. alkylphenol ethers like Triton™ X-100 with varying numbers of ethylene oxide residues render the determination difficult. Concerns over risks associated with bovine spongiform encephalopathy (BSE) have led to the introduction of a vegetable-derived form of polysorbate 80 manufactured from plant fats instead of bovine tallow. Despite their equal virucidal effectivity these polysorbates with a presumably different fatty acid profile may diverge in analytical sensitivity.
An early colorimetric assay based on the formation of a complex between polysorbate 80 and starch and measurement of the excess free starch by its reaction with iodine has been adapted for tissue culture media and vaccines containing amino acids, sugars, and protein. Another colorimetric assay based on the complexation of polyoxyethylene chains with thiocyanatocobaltate(II) and extraction of the complex into chloroform has originally been developed to determine the concentration of polyethylene glycol fatty acid esters in aqueous solutions. Since then, its use has been extended to other polyethoxylated nonionic surfactants such as e.g. p-isooctylphenol polyoxyethylene ethers (available from various manufacturers under the trade names Triton™ X-100, Igepal™ CA-630, Nonidet™ P40, or Tergitol™ NP40), with diethyl ether, methylene chloride or chloroform as extractant.
The application of this method for biologicals is however limited by the presence of proteins, which strongly interfere and, therefore, have to be removed before performing the analysis. To overcome protein-related matrix effects, separation techniques were introduced such as a cold ethanol precipitation step for protein removal by centrifugation before thiocyanatocobaltate(II) colorimetry, size-exclusion chromatography with post-column colorimetric derivatization, size-exclusion chromatography with a base concentration of polysorbate 80 above the critical micellar concentration to prevent micellar aggregation and UV detection at 235 nm, protein-depletion chromatography of unbound polysorbate with capture of protein on an ion-exchange HPLC column, solid-phase extraction with delipidation, colorimetric derivatization and separation of the thiocyanatocobaltate(II) complex by gel permeation chromatography, acidic hydrolysis with HPLC or GC determination of the fatty acid, or mild saponification with HPLC determination of the released fatty acid, thin-layer chromatography, or liquid extraction with HPLC separation and mass spectrometric detection. The latter approaches, which rely on the fatty acid moiety of polysorbate, are impeded by varying fatty acid compositions of the analyte. Another recent approach measures fluorescence polarization of 5-dodecanoylaminofluorescein incorporated into polysorbate micelles. However, these methods are either very time-consuming, suffer from limited sample throughput, or require complex instrumentation and separate validation of each step.
Therefore, a strong need exists to provide a method for the determination of polysorbate in protein-containing samples by a colorimetric assay, wherein the interference of protein can be eliminated in a fast and simple manner. Such a method should allow for the determination of the actual detergent concentration during e.g. virus inactivation, as well as in the final concentrates of purified proteins.
This need is satisfied by providing the embodiments characterized in the claims.